Our overall goal in this project is to characterize the functional contributions of Cdk5-mediated neuronal phosphorylation to the nervous system. Neuronal phosphorylation plays a crucial role in neuronal development and function, such as sensory recognition, behavioral control, memory, and learning. Most importantly, recent evidence suggests that abnormal phosphorylation in neurons is involved in the pathogenic mechanisms underlying many neurological diseases and drug addiction. Our main focus has been directed toward elucidating the contribution of cyclin-dependent kinase 5 (Cdk5) to brain development and function. Cdk5, a member of a large family of proline-directed serine/threonine protein kinases, was originally cloned by homology to other members of the Cdk family that are major regulators of cell-cycle progression. However, the involvement of Cdk5 in cell-cycle regulation has never been identified. Instead, it appears to play a major role in the nervous system. The neuronal specificity of Cdk5 kinase activity is achieved through the association with its activators, either p35 or p39, which are predominantly expressed in postmitotic neurons. To date, about 30 proteins with diverse functions have been identified as substrates of Cdk5, implicating it in the regulation of a wide range of neuronal functions. Apart from the critical role of Cdk5 in normal brain functions, deregulation of Cdk5 may be involved in the pathogenesis of neurodegenerative diseases such as Alzheimer?s disease, amyotrophic lateral sclerosis (ALS), or Parkinson?s disease, or in drug addiction due to a repeated exposure to cocaine. We have attempted to address the following questions in Cdk5 biology: ? What are the molecular mechanisms underlying the migration defects of Cdk5-deficient neurons? ? What is the functional significance of the coexistence of two Cdk5 activators, p35 and p39? ? Is Cdk5 required for the proper development of brainstem structures, including facial nuclei? ? Is there ?crosstalk? of Cdk5 with other molecules involved in neuronal migration? ? What are the functions of Cdk5 in mature neurons (e.g., synaptic or post-synaptic signal transmission)? ? Does Cdk5 contribute to neuronal survival in physiological or pathogenic conditions? We have used a variety of functional genomic techniques and molecular approaches, including conventional and conditional gene targeting, analysis of protein-protein interactions, as well as behavioral tests in mice to address these questions. We believe our present studies will not only contribute to a greater understanding of the molecular basis of Cdk5 functions in the brain, but also will help future efforts toward the development of more effective diagnosis and treatments for debilitating neurodegenerative and addictive diseases. Cdk5-dependent migration of facial branchiomotor (FBM) and inferior olive (IO) neurons: Our initial analysis of Cdk5-null mice revealed that disruption of Cdk5 expression resulted in an abnormal layering structure of the cerebrum and cerebellum, which was caused by migration defects of the postmitotic neurons. The molecular mechanism underlying the migration defects was further examined using chimeric mice generated by injection of Cdk5-null ES cells into host blastocysts. In these chimeric mice, only the neurons derived from the Cdk5-null ES cells failed to migrate, indicating the cell autonomous nature of the defect of Cdk5-deficient neurons. Our research was further extended to search for Cdk5-dependent migration in other brain areas, and we identified novel migration defects of FBM and IO neurons in Cdk5-null mice. The migration of Cdk5-deficient FBM neurons was completely arrested in the dorsal pons where they were born, resulting in their failure to migrate into the ventral pons. The specificity of ectopic FBM neurons was ascertained by the expression of Phox2b, peripherin, and cadherrrin-8,, which are known to be expressed in wild-type FBM neurons. Moreover, in the hindbrain of Cdk5-null mice, the IO neurons failed to form the distinct structures of the inferior olive, while they migrated close to their final destination from the lower rhombic lip. Based on these findings, we have grouped the defects of neuronal positioning caused by Cdk5 deficiency into three different categories. The first category contains the FBM neurons and Purkinje cells, which did not migrate from their sites of origin. The second category includes the IO neurons and mitral cells in the olfactory bulb, which failed to properly align. The third category contains neurons with an intermediate defect: later-born neurons of the cerebral cortex may be placed in this intermediate group, in which Cdk5-deficient cortical neurons were unable to migrate past their predecessors, leading to an inversion of cortical layers. Therefore, we believe that the precise delineation of Cdk5-dependent migration will provide valuable insight into the molecular and cellular mechanisms of neuronal migration during brain development. Critical role of Cdk5 in neuronal survival: Cdk5-null mice showed a unique phenotype, characterized by perinatal lethality and abnormal cytoarchitecture of the brain. In addition, neurons in the brain stem and spinal cord exhibited chromatolytic changes with an accumulation of neurofilaments, which are known as pathological hallmarks of some neurodegenerative diseases. It had not been clear, however, whether these phenotypes in Cdk5-null mice could be primarily attributed to defects in neurons or in glial cells, because Cdk5 is expressed in both neurons and glial cells. To address this question, we have generated a double-transgenic mouse in which the Cdk5 transgene was expressed in an endogenous Cdk5-null background under the control of the p35 promoter. This reconstitution of Cdk5 expression only in neurons led to a complete rescue of the phenotype observed in Cdk5-null mice, suggesting that the phenotype of Cdk5-null mice is likely to stem from defects in neurons. We have found that apoptotic cell death is prevalent in the brain of Cdk5-null mice and that Cdk5-deficient neurons display a higher sensitivity to UV irradiation. However, the mechanism by which Cdk5 controls neuronal survival remains to be determined. Members of the c-Jun NH2-terminal kinase (JNK) family are known as stress-activated protein kinases. Mice lacking JNK3 exhibited a resistance to kainic acid-induced apoptosis in the hippocampus, indicating the preferential role of JNK3 in stress-induced neuronal apoptosis. These data led us to examine a link between JNK3 and Cdk5 in regulating neuronal apoptosis. We found that Cdk5 phosphorylated JNK3 on Thr-131 and inhibited its activity. Actually, Cdk5-null mice exhibited an increased phosphorylation of c-Jun, a target molecule of JNK3. Based on these findings, we propose that Cdk5 might play a role in neuronal survival by negatively regulating JNK3. Regulation of Cdk5 and tau phosphorylation: Cdk5-null mice displayed extensive defects in all brain areas, while p35-null mice displayed defects mostly confined to the forebrain. This difference in phenotypic severity suggested the functional importance of the other Cdk5 activator, p39, in brain development. Therefore, we hypothesized that the coexistence of Cdk5 activators p35 and p39 might contribute to the in vivo activation of Cdk5 in a region-specific or developmentally regulated manner. We examined p35 and p39 expression levels in the developing mouse brain. Our data clearly demonstrated that p39 expression was higher during embryonic development in the cerebellum, brain stem, and spinal cord, where the pathological defects were mostly absent in p35-null mice. However, the level of p39 expression was very low in the embryonic cerebral cortex, where p35-null mice had lamination.